The present application is the national stage under 35, U.S.C. 371, of international application PCT/JP00/08465, filed Nov. 30, 2000, which designated the United States, and which international application was not published under PCT Article 21(2) in the English language.
The present invention relates to a precast polyacrylamide gel adapted for use in electrophoresis separation, production method thereof and electrophoresis using the gel.
Precast polyacrylamide gels for electrophoresis are extensively used as a basic investigative medium for detecting and quantitatively analyzing chemical substances: proteins, nucleic acids, carbohydrates, and lipids necessary for building all living organisms in fields as diverse as biology, medicine, fisheries, veterinary medicine, and so on. Especially, a variety of precast polyacrylamide gels different in resolving power can be made easily by varying a gel recipe because the gels are substances synthesized artificially. Accordingly, it is possible to prepare in advance the precast gels differing diversely in separation characteristic or resolution from one another. The artificial gels, since much saving the labor and effort for analytic procedure and excellent in uniformity and reproducibility, have helped the productivity and quality control in the fields stated earlier. The artificial gels which are mass-produced must have an increased shelf-life to ensure an adequate supply of these gels.
Most polyacrylamide gels for electrophoresis used extensively for analytic technique of proteins in the fields of biochemistry and medicine are based on the system developed by Ornstein [L. Ornstein, Ann. N.Y. Acad. Sci. 121, 321-349, (1964)] and Davis [B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404-427(1964)] or that proposed by Laemmli [U. K. Laemmli, Nature, 227, 680 (1970)]. Gels have heretofore been produced by the users or analysts in themselves or on themselves for their private use. In particular, the Laemmli system is most extensively used, since the molecular weights of proteins can be simply presumed by adding sodium dodecyl sulfate, contracted to xe2x80x9cSDSxe2x80x9d hereinafter, to gels or electrophoresis buffer solutions. The Laemmli system consists of a gel buffer solution of tris(hydroxymethyl) aminomethane, contracted to xe2x80x9cTrisxe2x80x9d herein, which is partially neutralized with hydrochloric acid, and an electrophoresis buffer solution, or Laemmli""s electrophoresis buffer solution using Tris and glycine salt. The gel buffer solution in the system stated earlier, or Laemmli""s gel buffer solution contains about 10 -20 mol % Tris partially neutralized to be adjusted to pH 8.8 with hydrochloric acid. At this pH of the Laemmli""s gel buffer solution, however, amide groups are subjected to hydrolysis time elapses. As the hydrolysis of gels proceeds even under low temperatures, the polyacrylamide gels come to contain partially anionic groups. As a result, the migration distances of proteins are reduced while electrophoretic patterns become vague, and therefore it is impossible to preserve the gels over a prolonged period.
The following discussions will present the electrophoresis of nucleic acids. Recently remarkable development of molecular biology has come to need the analysis and preparation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The analysis of nucleic acids mostly depends on either the agarose electrophoresis separation process or the precast polyacrylamide gel electrophoresis separation process. As nucleic acids present strong negative charges in the neutral buffer solution while the mobility thereof depends on the molecular sieve effect of supportive matrix gels, either an about 0.3-2% agarose gel or an about 3.5-20% polyacrylamide gel is chiefly employed depending on the size of nucleic acids undergoing analysis.
The agarose gel, because of having a larger pore size, is often used for analytic separation of macromolecular nucleic acids. There are many types of agarose gels, which differ from one another in electroosmosis, gel strength, melting point, and so on. Moreover, because the agarose gels are naturally occurring materials, the differences in quality are frequently seen., even for a lot of products and even for the same gels produced in the same facility. For purifying DNA by virtue of the agarose gel electrophoresis separation process, any other purification procedures such as the phenol extract, and so on must be considered because contamination with impurities in the agarose gels would inhibit enzyme activities of restriction enzymes, DNA polymerases, DNA synthetase, and so on.
In contrast, polyacrylamide gels with relatively small pore size are useful for isolation and analysis of nucleic acids of medium and/or low molecular weight. Because the polyacrylamide gels are artificially synthetic products, they are very pure chemically, and thus involving no major problems seen in the agarose gels. Moreover, the polyacrylamide gels may be easily prepared in diverse types of gels, which are different in separation characteristics, depending on the desired gel recipe. Accordingly, it is possible to prepare in advance the precast gels differing diversely in separation characteristics from one another. The use of artificial gels mass-produced in advance diverse separation characteristics, since much saving the labor and effort for analytic procedure and excellent in uniformity and reproducibility, will help productivity and quality control in the fields stated earlier. The artificial gels obtained by mass-production methods have increased shelf-life because of preservation. The most common buffer system for using agarose and polyacrylamide as the gel material in the electrophoresis separation of DNA is a continuous buffer system of around pH 7.8 to 8.3, in which a gel buffer solution is equal in composition with an electrophoresis buffer solution: a tris-acetic acid buffer, contracted to xe2x80x9cTAExe2x80x9d herein, which contains either ethylenediaminetetraacetic acid, contracted to xe2x80x9cEDTAxe2x80x9d or disodium ethylenediaminetetraacetate, contracted to xe2x80x9cETAxe2x80x9d; a tris-boric acid buffer, abbreviated to xe2x80x9cTBExe2x80x9d; and a tris-phosphoric acid, abbreviated to xe2x80x9cTPExe2x80x9d.
As stated earlier, the precast gels manufactured on mass-production methods must be able to maintain the long-lasting shelf-life in preservation throughout shipping and storage. Among the prior processes for manufacturing gels which are superior in shelf-life is a process for manufacturing a polyacrylamide gel having the increased shelf-life disclosed in Published Unexamined Patent Application in Japan No. H04-184 163, which involves a gel buffer solution composed of Tris, ampholyte and acid to be storable for a prolonged period without loss of performance and also expanded remarkably in molecular weight range applicable for analytical electrophoresis. The polyacrylamide electrophoresis gel produced by the process recited just above is available for analysis using Laemmli""s electrophoresis buffer solution. The polyacrylamide gel contains the ampholyte and has the pH of ranging from 4.0 to 7.5. In an example disclosed, the gel having a pH of ranging from 6.9 to 7.4 is produced, which is described to be stored under refrigeration for four months without variation in mobility of proteins.
The gel buffer solution is set high in neutralization rate of tris to depress the hydrolysis of the polyacrylamide gel, while added with the ampholyte to make gentle a variation in electric potential gradient neighboring a boundary between tris strong acid salt part and tris week acid salt part, thereby coming to exhibit the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using the Laemmli""s electrophoresis buffer solution.
Nevertheless, most polyacrylamide gels containing a gel buffer solution of the pH range 6.9 to 7.4 begin to vary in electrophoretic mobility of proteins after more than five months in storage under refrigeration has gone by. Moreover, the polyacrylamide gel in the form of slab gel is apt to change in shape of gel, tending to dislocate the glass plates. This results in major problems of rendering handling procedure on the electrophoretic analysis worse, causing the cracking of the gels due to the dislocation of the glass plates, and so on. Although it will be speculated that the gels may be improved in shelf life with a gel buffer solution of pH 6.8 or less, such gels have disadvantages of being time-consuming for electrophoretic migration and also reducing in the resolution. Thus, none could not have succeed in developing a polyacrylamide gel that can be stored for over five months without loss of performance, superior in the resolution for proteins, and also exhibit the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using the Laemmli""s gel buffer solution.
In addition, International Publication Number WO 95/27 197 discloses a buffer system for a long-lasting precast electrophoresis gel wherein separation occurs at neutral pH. The gel buffer solution in the buffer system contains a mono organic amine or substituted amine with a pH near neutrality, titrated with hydrochloric acid to a pH of between about pH 6 and about pH 7. The electrophoresis buffer solution in the buffer system recited above contains a zwitterion selected from the group of MOPS, MES, ACES, MOPSO, TES, HEPES and TAPSO, titrated to a pH of about pH 7 with sodium hydroxide or organic base. Both the gel buffer solution and the electrophoresis buffer solution are kept at neutral pH and in doing so the polyacrylamide gel itself is less subject to hydrolysis while proteins are separated with remaining completely reduced. The electrophoresis gel system recited earlier is described to have an increased useful shelf-life up to twelve months.
Nevertheless, the buffer system disclosed in International Publication Number WO 95/27 197 requires using special molecule markers, or molecules of known molecular weights. Moreover, the polyacrylamide in International Publication Number WO 95/27 197 gets slower in the rate of electrophoretic migration when using any prior electrophoresis buffer solution, for example a commonly available Laemmli""s electrophoresis buffer solution containing glycine. Thus, it is impossible to use polyacrylamide for analytic separation of proteins.
The polyacrylamide controlled to a pH ranging from neutral to acid, which is considerably resistant to hydrolysis, may serve well as the electrophoresis gel improved in shelf life. That is, the polyacrylamide set at a pH ranging from neutral to acid is prolonged in shelf life, and thus may be produced in advance by mass production methods to have diverse separation characteristics. If the polyacrylamide gel combined with the precast gel technology makes it possible to separate analytically DNA by the use of the electrophoresis buffer solution available for the electrophoresis of proteins, and also to separate analytically DNA by the use of the electrophoresis buffer solution commonly available for the electrophoresis of nucleic acids, it will be expected to realize the electrophoretic analysis with efficiency at low costs.
A primary object of the present invention to solve the subject matter stated earlier is to provide a precast polyacrylamide gel that can be stored under refrigeration over more than six months with preserving the gel shape without loss of resolution of proteins, while coming to exhibit the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using the Laemmli""s electrophoresis buffer solution.
A second object of the present invention to solve the subject matter stared earlier is to provide an electrophoresis method of separating and analyzing distinctly DNA by the use of a polyacrylamide electrophoresis gel storable under refrigeration over more than six months without loss of performance, together with an electrophoresis buffer solution availed commonly for the analytic separation of nucleic acids.
A third object of the present invention to solve the subject matter stated earlier is to provide an electrophoresis method of separating and analyzing distinctly DNA by the use of a polyacrylamide gel available for separation of proteins and storable under refrigeration over more than four months without loss of performance, together with an electrophoresis buffer solution for the analysis of proteins.
In an aspect of the present invention, a precast polyacrylamide gel adapted for use in gel electrophoresis is disclosed, in which an electrophoresis buffer solution is composed of an aqueous solution containing a tris(hydroxymethyl) aminomethane and an ampholyte, and wherein the tris(hydroxymethyl) aminomethane is present in a concentration of between 0.07 mol/litre and 0.2 mol/litre while the ampholyte is composed of glycine and at least one conjugate ampholyte, which has a basic dissociation constant of 8.3 pKb 9.6, the conjugate ampholyte added ranging in amount from 0.1 to 30 mol % with respect to the glycine, concentrations of the conjugate ampholyte and the glycine ranging in all between 0.1 and 0.3 mol/litre, and a pH range being between pH 6.0 and pH 6.8.
The precast polyacrylamide gel of the present invention can be stored under refrigeration for over six months without loss of resolution and gel configuration, and also exhibit the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using the Laemmli""s electrophoresis buffer solution.
In another aspect of the present invention, there is disclosed that the conjugate ampholyte is an amino acid having the same number of anionic and cationic groups in a molecule.
In a further another aspect the present invention, there is disclosed that the conjugate ampholyte is at least one selected from serine, glutamine, tryptophan, methionine and phenylalanine.
In another aspect of the present invention, a process for fabricating a precast polyacrylamide gel suitable for electrophoresis is disclosed, which is comprised of polymerizing an admixture of an acrylamide, polyfunctional crosslinking agent, water and a buffer solution of composition defined in the following (1) and (2) in the presence of a polymerization initiator under the condition of pH range 6.0 6.8:
(1) a tris(hydroxymethyl) aminomethane is present in a concentration of between 0.07 mol/litre and 0.1 mol/litre; and
(2) an ampholyte is composed of glycine and at least one conjugate ampholyte,
(a) the conjugate ampholyte has a basic dissociation constant of 8.3 pKb 9.6,
(b) the conjugate ampholyte added ranges in amount from 0.1 to 30 mol % with respect to the glycine, and
(c) a total concentration of the conjugate ampholyte and the glycine ranges between 0.1 and 0.3 mol/litre.
According to the process for fabricating a precast polyacrylamide electrophoresis gel as stated earlier, the improved precast polyacrylamide electrophoresis gel may be obtained with efficiency, which is increased in the useful shelf life where the gel can be stored for over six months without loss of resolution and gel configuration.
In another aspect of the present invention, a use of the precast polyacrylamide electrophoresis gel is disclosed, in which the analytic separation of proteins is performed using the electrophoresis buffer solution containing glycine and tris(hydroxymethyl) aminomethane with added dodecyl sulfate.
The use of the precast polyacrylamide electrophoresis gel as stated just above makes it possible to realize the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using the Laemmli""s gel electrophoresis buffer solution.
In a further another aspect of the present invention, an electrophoresis system for DNA is disclosed, which makes use of an electrophoresis buffer solution containing tris(hydroxymethyl) aminomethane and a precast polyacrylamide gel characterized by the following (1) and (2);
(1) a gel buffer solution contained in the precast polyacrylamide gel contains tris(hydroxymethyl) aminomethane, at least one ampholyte rendering glycine indispensable, and a monobasic acid, titrated to a pH of between pH 6.0 and pH 7.5, and
(2) the ampholyte contained in the gel buffer solution has a basic dissociation constant of 8.3 pKb 9.6, and includes an amino acid having the same number of anionic and cationic groups in a molecule.
In another aspect of the present invention, the gel buffer solution contains a monobasic acid consisting of at least one of hydrochloric acid and acetic acid, titrated to a pH of between pH 6.0 and pH 6.5 to increase the useful shelf life of a precast gel.
In another aspect of the present invention, the gel buffer solution contains a monobasic acid consisting of at least one of hydrochloric acid and acetic acid, titrated to a pH of between pH 6.5 and pH 7.5 to increase the useful shelf life of a precast gel.
In another aspect of the present invention, an electrophoresis buffer solution is disclosed which contains, aside from tris(hydroxymethyl) aminomethane, components defined in the following (1) and (2), titrated to a pH in a pH range 7.8 8.3:
(1) any one of acetic acid, phosphoric acid and boric acid; and
(2) a chelating agent of disodium ethylenediaminetetraacetate.
In a further another aspect of the present invention, there is disclosed the electrophoresis buffer solution containing the monobasic acid of boric acid.
In another aspect of the present invention, there is disclosed the electrophoresis buffer solution containing an ampholyte, aside from tris(hydroxymethyl) aminomethane, and titrated to a pH in a pH range 7.8 8.3.
In another aspect of the present invention, there is disclosed the electrophoresis buffer solution containing the ampholyte of glycine.
In a further another aspect of the present invention, there is disclosed the electrophoresis buffer solution containing dodecyl sulfate, in addition to the ampholyte of glycine.
In accordance with the present invention, a gel adapted for use in analytic separation of proteins is provided, which is enhanced in the useful shelf-life thereof. The gel contains therein a buffer solution composed of Tris, glycine and at least one conjugate ampholyte, the Tris ranging in concentration in the gel from 0.07 mol/litre to 0.2 mol/litre, preferably from 0.08 mol/litre to 0.2 mol/litre. The limited concentration range recited above results in inclusion of leading ions enough in amount to ensure the electrophoresis time comparable to the gels fabricated using the Laemmli""s gel buffer solution. With the Tris less in concentration in the gel than 0.07 mpl/litre, the electrophoretic pattern becomes overall too vague to avail for the analytic separation of proteins. In contrast, even if the concentration of the Tris in the gel is over 0.2 mol/litre, the concentration of the leading ion in the gel becomes too high, thereby prolonging the electrophoresis time, making the operational efficiency of the analysis worse.
The ampholyte is composed of glycine and at least one conjugate ampholyte that has the basic dissociation constant of 8.3 pKb 9.6, preferably 9.0 pKb 9.6. Only glycine by itself, especially when the gel buffer solution is at pH 7 or less, can not realize the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using the Laemmli""s gel buffer solution. On the other hand, with the conjugate ampholyte of pKb 8.3, the ampholyte is lower in pKb than the Tris, and for the sake of which the gel buffer solution is varied in pH to make the fractionated molecular weight range much broaden. In contrast, if the conjugate ampholyte is in the condition of pKb 9.6, the ampholyte exceeds the glycine in basic dissociation constant, making no contribution to helping ensure the gentle potential gradient. To cope with this, when the conjugate ampholyte is selected from the group of serine, tryptophan, phenylalanine, and so on satisfying 9.0 pKb 9.6, the ampholytes migrate consecutively towards the anode side to their respective positions on the basis of the pKb value, with the smallest pKb migrating to the position most neighboring the anode, so that the gentle potential gradient may be established in the gel. Thus, the precast polyacrylamide gel of the present invention succeeds in realizing the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using the Laemmli""s gel buffer solution.
According to the present invention, the amount of the conjugate ampholytes added is from 0.1 to 30 mol %, preferably from 1 to 20 mol %, with respect to glycine. When the conjugate ampholyte added is less in amount than 0.1 mol %, the precast polyacrylamide gel fails in realizing the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using the Laemmli""s gel buffer solution. To the contrary, if the conjugate ampholyte is added over 30 mol %, the electrophoretic image appearing on the gel becomes too vague to have any practical use.
The total concentration of the conjugate ampholyte and glycine is in the range of from 0.1 mol/litre to 0.3 mol/litre, preferably from 0.1 to 0.2 mol/litre. Even when the ampholyte is either less than 0.1 mol/litre or more than 0.3 mol/litre, the electrophoretic pattern on the gel also becomes vague to be unavailable for practical use thereof.
The ampholytes used in the present invention preferably have the same number of anionic and cationic groups in a molecule. All the anionic and cationic groups dissociate into ions in the gel buffer solution so that the ampholytes become electrically neutral. At a pH higher than pKb, only the anionic groups dissociate while the cationic groups are less subject to the dissociation so that the ampholytes is charged negatively. Thus, it will be understood that the ampholytes behave substantially like weak monobasic acid.
The given pH of the gel buffer solution for the precast polyacrylamide gel of the present invention, the use and production thereof is adjusted to from 6.0 to 6.8, preferably to 6.3. When the buffer solution is above pH 6.8, the polyacrylamide is susceptible to hydrolysis, and thus can not be stored for a long period without loss of performance. In contrast, if the gel buffer solution is less than 6.0 in pH, the polyacrylamide gel may keep the increased shelf-life while the electrophoretic patterns of proteins are liable to become too vague to have the practical use. The polyacrylamide gel with the gel buffer solution adjusted to pH 6.8 can be stored under refrigeration over six months with no loss of performance. When controlled to just pH 6.3, the gels obtained are remarkably retarded in the rate of hydrolysis compared with the pH 6.8, because the pH 6.3 is near to the pH of the acrylamide itself. This makes it possible to store the gels for over a year with no change or deterioration in gel shape and electrophoretic pattern due to acrylamide hydrolysis.
The use of the precast polyacrylamide gels according to the present invention is an analytic separation of proteins, which employs the electrophoresis buffer solution containing Tris and glycine, preferably containing Tris of 0.025 mol/litre, glycine of 0.192 mol/litre and SDS of 0.1% by weight. The electrophoresis buffer solution, because of having the same composition found in the extensively used Laemmli""s gel buffer solution, serves well for the analytic separation of proteins with inexpensive cost and efficiency. Selection of an ampholyte, for example MOPS rather than glycine for the electrophoresis buffer solution may also serve for the analytic separation of proteins. Nevertheless, the fractionated molecular weight range performed by the gel is broader, compared with the gel fabricated using the Laemmli""s gel buffer solution used extensively.
The precast polyacrylamide gel of the present invention may be produced in the same fashion as the production of the prior polyacrylamide electrophoresis gels, excepting pH regulation and addition of ampholytes. For instance, N,Nxe2x80x2-methylene bis-acrylamide, contracted to xe2x80x9cBISxe2x80x9d herein, is among the most extensively used water-soluble divinyl compounds, which are able to conjugate with the acrylamide for cross-linking the acrylamide. Any other suitable cross-linker may be utilized such as N,Nxe2x80x2-diallyltartardiamide, which is a kind of the commonly available water-soluble divinyl compounds.
An aqueous monomer solution used in the precast polyacrylamide gel of the present invention may include a water-soluble polymer such as agarose, polyacrylamide, polyvinyl alcohol, polyvinyl pyrollidone, polyethylene oxide, polymethyl vinylether, and so on to endow the gel with elasticity and increase the gel strength. Alternatively, the monomer solution may be copolymerized with other monomers.
Copolymerization of monomers in the production of the precast polyacrylamide gel of the present invention takes place with radicals resulting from either polymerization initiator or exposure to any of ultraviolet light and ionizing radiation. The polymerization initiator is preferably, but limited to a polymerization initiator of redox system in which a peroxide such as ammonium persulfate, and so on is simultaneously used with a reducing agent such as N,N,Nxe2x80x2,Nxe2x80x2-tetramethylethylenediamine, contracted to TEMED herein, and so on. The peroxide and reducing agent are used in the range of from 0.05 to 5% by weight/volume based on the total monomer. Copolymerization temperature, although unspecified as long as the initiator is allowed to serve well, is preferably in the range of from 15 to 50.
The following description will present the electrophoresis separation for DNA in which the precast polyacrylamide gel constructed according to the present invention to have the increased shelf-life is used in combination with the electrophoresis buffer buffer solution commonly used for the analytic separation of nucleic acid.
The gel buffer solution contains Tris, any one or more ampholytes including glycine, and a monobasic acid. Preferably, the gel buffer solution contains Tris, glycine, an amino acid having the same number of anionic and cationic groups in a molecule of 8.3 pKb 9.6 in basic dissociation constant, hydrochloric acid and acetic acid. The gel buffer solution without any one or more than two of ampholytes including glycine, when made acidic in pH, gets Tris to rise in neutralization rate, causing much more variation in the potential gradient at an area neighboring a boundary between the tris strong acid salt part and the tris week acid salt part. This is apt to incur focusing of separated molecules near the boundary area, rendering the fractionated molecular weight range much reduced. Besides, the electrophoresis time is prolonged so that the separation operation is intolerably retarded in efficiency.
Since glycine may serve well to make gentle the change in potential gradient at the area near the boundary between the tris strong acid salt part and the tris week acid salt part in the electrophoresis gel, it is inevitable for realizing the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using either the TBE or the Ornstein and
Davis gel buffer solution. Nevertheless, even if the gel buffer solution is made much acid in pH, glycine can not control the change in potential gradient alone. To cope with this, it is desired in the gel buffer solution to conjugate glycine with any amino acid having the same number of anionic and cationic groups in a molecule of 8.3 pKb 9.6 in basic dissociation constant. This makes it possible to render the potential gradient in the electrophoresis gel gentle.
The amino acids conjugated with glycine have the basic dissociation constant pKb of 8.3 pKb 9.6, more preferably of 9.0 pKb 9.6. In case where the amino acids conjugated with glycine has pKb 8.3, the amino acids are lower in pKb than the Tris, and for the sake of which the gel buffer solution is varied in pH to make the fractionated molecular weight range much broadening. Conversely, when glycine is conjugated with the amino acids of pKb 9.6, the amino acids exceed the glycine in pKb, making no contribution to helping ensure the gentle potential gradient. Where the amino acids of 9.0 pKb 9.6 are used in the gel buffer solution, they migrate consecutively towards the anode side to their respective positions on the basis of the pKb value, with the smallest pKb migrating to the position most neighboring the anode, so that the gentle potential gradient may be established in the gel. Among the amino acids satisfying 9.0 pKb 9.6 are serine, tryptophan, phenylalanine, and so on. According to the present invention, serine is preferred.
Moreover, it is desirable that the amino acids conjugated with glycine have the same number of anionic and cationic groups in a molecule. At a pH somewhat lower than pKb, all the anionic and cationic groups dissociate completely into ions so that the amino acids takes on electric neutrality. In contrast, at a pH higher than pKb, only the anionic groups dissociate whereas the cationic groups are retarded in dissociation, so that the amino acids are charged negatively. Thus, the amino acids exhibit substantially the behavior as monobasic weak acid to be allowed to use them as quasi-weak acid. Previously adding any quasi-weak acid to the gel buffer solution results in lowering an electric resistance of the tris-weak acid salt part in the electrophoresis gel. Under the presence of strong acid radicals, in addition, the pH is so low that the amino acids become electrically neutral, thus making no contribution to electric conduction.
The pH of the gel buffer solution is in the range of from 6.0 to 7.5, preferably from 6.0 to 6.5. With the pH above 7.5, the polyacrylamide gels, because of being subjected to hydrolysis over time, are limited in shelf-life thereof. Conversely, the pH below 6.0, although increasing the useful shelf-life of the polyacrylamide gels, raises the neutralization rate of Tris to an extent that might impair the effects of the buffer system. In order to increase the useful shelf-life of the precast polyacrylamide gels, it is preferable to get the pH of the gel buffer solution to near the pH of the acrylamide per se.
On the other hand, the electrophoresis buffer solution contains Tris, chelating agent, and any one acid of acetic acid, phosphoric acid and boric acid, preferably boric acid. With the gel buffer solution adjusted to a pH in the pH range of from 6.0 to 6.5, the Ornstein and Davis electrophoresis buffer solution is not practical for the analytic separation of DNA because the electrophoretic patterns obtained become vague. The TBA employed in the present invention contains Tris of 0.0892 mol/litre, boric acid of 0.0890 mol/litre and ETA of 0.0025 mol/litre, preferably titrated to pH 8.3. The buffer system of the composition stated just earlier is the TBE extensively used for analytic separation of nucleic acid, and therefore may serve well for the analysis with inexpensive cost and efficiency. Moreover, the buffer system stated earlier makes it possible to finish the electrophoresis within a limited short period of time while ensuring very clear patterns. Either of TAE and TPE, although enabling the analytic separation of DNA, needs plenty of time to conduct the electrophoresis while becoming worse in the analytic efficiency, compared with the TBE.
The following description will present the electrophoresis separation for DNA in which the precast polyacrylamide gel constructed according to the present invention to increase in the useful shelf-life is used in combination with the electrophoresis buffer solution commonly used for the electrophoresis of proteins.
The gel buffer solution contains Tris, any one or more than two of ampholytes including glycine, and a monobasic acid. Preferably, the gel buffer solution contains Tris, glycine, an amino acid having the same number of anionic and cationic groups in a molecule of 8.3 pKb 9.6 in basic dissociation constant, hydrochloric acid and acetic acid. The gel buffer solution without any one or more than two of ampholytes including glycine, when made acidic in pH, gets Tris to rise in neutralization rate, causing much more variation in the potential gradient at an area neighboring a boundary between the tris strong acid salt part and the tris weak acid salt part. This is apt to incur focusing of separated molecules undergoing analysis nearby the boundary area, rendering the fractionated molecular weight range much reduced. Besides, the electrophoresis time is prolonged so that the separation operation is intolerably retarded in efficiency.
Since glycine may serve well to make gentle the change in potential gradient at the area near the boundary between the tris strong acid salt part and the tris weak acid salt part even in the low pH gel buffer solution, the existence of glycine in the gel buffer solution is inevitable for realizing the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using either the TBE or the Ornstein and Davis gel buffer solution. Nevertheless, when the gel buffer solution is made more acid in pH, glycine can not control the change in potential gradient alone. To cope with this, it is desired in the gel buffer solution to conjugate glycine with any amino acid having the same number of anionic and cationic groups in a molecule of 8.3 pKb 9.6 in basic dissociation constant. This makes it possible to render the potential gradient in the electrophoresis gel gentle.
The amino acids to be conjugated with glycine have the basic dissociation constant pKb of 8.3 pKb 9.6, more preferably of 9.0 pKb 9.6. In case where the amino acids conjugated with glycine has pKb 8.3, the amino acids are lower in pKb than the Tris, and for the sake of which the gel buffer solution is varied in pH to make the fractionated molecular weight range much broadening. Conversely, when glycine is conjugated with the amino acids of pKb 9.6, the amino acids exceed the glycine in pKb, making no contribution to helping ensure the gentle potential gradient. Where the amino acids of 9.0 pKb 9.6 are used in the gel buffer solution, they migrate consecutively towards the anode side to their respective positions on the basis of the pKb value, with the smallest pKb migrating to the position most neighboring the anode, so that the gentle potential gradient may be established in the gel. Among the amino acids satisfying 9.0 pKb 9.6 are serine, tryptophan, phenylalanine, and so on. According to the present invention, serine is preferred.
Moreover, it is desirable that the amino acids conjugated with glycine have the same number of anionic and cationic groups in a molecule. At the pH somewhat lower than pKb, all the anionic and cationic groups dissociate completely into ions so that the amino acids takes on electric neutrality. In contrast, at the pH higher than pKb, only the anionic groups dissociate whereas the cationic groups are retarded in dissociation, so that the amino acids are charged negatively. Thus, the amino acids exhibit substantially the behavior as monobasic weak acid to be allowed to use them as quasi-weak acid. Previously adding any quasi-weak acid to the gel buffer solution results in lowering an electric resistance of the tris-weak acid salt part in the electrophoresis gel. Under the presence of strong acid radicals, in addition, the pH is so low that the amino acids become electrically neutral, thus making no contribution to electric conduction.
The pH of the gel buffer solution is in the range of from 6.5 to 7.5, preferably from 6.5 to 7.0. With the pH above 7.5, the polyacrylamide gels, because of hydrolysis over time, are limited in shelf-life thereof. Conversely, a pH below 6.5, although increasing the useful shelf-life of the polyacrylamide gels, results in unsatisfactory electrophoresis patterns.
The electrophoresis buffer solution employable in the present invention is the Ornstein and Davis electrophoresis buffer solution containing 0.025 mol/litre Tris and 0.192 mol/litre glycine, titrated to the pH 8.3. The buffer solution composed as stated earlier is the electrophoresis buffer solution extensively used in the analytic separation of proteins and, therefore, may serve well with good efficiency to obtain very clear electrophoresis results.
For the electrophoresis buffer solution of the present invention, alternatively, the Laemmli""s electrophoresis buffer solution commonly used in the protein analyses may be also adopted, which contains 0.025 mol/litre Tris, 0.192 mol/litre glycine and 0.1%(w/v) SDS. Thus, both the analytic separations of DNA and proteins may be run on the same electrophoresis buffer solution. This contributes to the efficient operation and cost reduction.
While the present invention will be explained with reference to the following examples, it should be understood that such examples are not restrictive, but are for illustrative purposes only.